Cacao black pod rot, caused by Phytophthora palmivora, is an economically serious problem in all cacao-producing regions, leading to global yield loss and tree deaths. With the advent of Republic Act 10068, also known as the Organic Agriculture Act of 2010, the state is searching for and encouraging sustainable agriculture. Hence, this study was conducted to determine the antagonistic effects of the different biological control agents against P. palmivora and identify the most effective biological control agents against cacao pod rot. Conducted at a laboratory, the experiment was laid out in Complete Randomized Design (CRD) and replicated three times with five samples of cacao pods per replication. Treatments used were as follows; T1- Negative control, T2- P. palmivora, T3- Chemical control, T4- Bacillus subtilis, T5- Bacillus amyloliquefaciens, T6- Trichoderma harzianum. The in vitro test was done using a dual culture technique, while the in vivo test was done through a detached pod test. The dual culture test showed that T. harzianum reduced P. palmivora radial growth to 6.77mm with a percent growth inhibition of 70.54%. Moreover, the detached pod test recorded no disease incidence, severity, and incubation period in pods treated with T. harzianum. Whereas, the longer incubation period was noted in pods treated with chemical control at 4.27 days. Moreover, the highest degree of control was recorded in pods treated with T. harzianum (100%). These findings emphasized the potential of T. harzianum as a sustainable and effective solution for combating cacao black pod rot and promoting sustainable agriculture.

1. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/378861059
Utilization of Biological Control Agents against Phytophthora palmivora
Causing Pod Rot Disease in Cacao
Article in Twist · March 2024
DOI: 10.5281/zenodo.10049652#109
CITATIONS
0
READS
39
3 authors, including:
Jeson Geroche
Davao de Oro State College
8 PUBLICATIONS 3 CITATIONS
SEE PROFILE
All content following this page was uploaded by Jeson Geroche on 11 March 2024.
The user has requested enhancement of the downloaded file.

2. 431
https://doi.org/10.5281/zenodo.10049652#109 TWIST, 2024, Vol. 19, Issue 1, pp. 431-438
T W I S T
Journal homepage: www.twistjournal.net
Utilization of Biological Control Agents against Phytophthora
palmivora Causing Pod Rot Disease in Cacao
Jeson N. Geroche*
Associate Professor, Agriculture Department, Davao de Oro State College, Philippines
[*Corresponding author]
Rey D. Macaya
Research Scholar, Agriculture Department, Davao de Oro State College, Philippines
Jebuchadnezzar L. Seprado
Instructor II, Institute of Agriculture and Related Sciences,
Kapalong College of Agriculture, Sciences and Technology, Philippines
Abstract
Cacao black pod rot, caused by Phytophthora palmivora, is an economically serious problem in all cacao-producing
regions, leading to global yield loss and tree deaths. With the advent of Republic Act 10068, also known as the Organic
Agriculture Act of 2010, the state is searching for and encouraging sustainable agriculture. Hence, this study was
conducted to determine the antagonistic effects of the different biological control agents against P. palmivora and identify
the most effective biological control agents against cacao pod rot. Conducted at a laboratory, the experiment was laid out
in Complete Randomized Design (CRD) and replicated three times with five samples of cacao pods per replication.
Treatments used were as follows; T1- Negative control, T2- P. palmivora, T3- Chemical control, T4- Bacillus subtilis,
T5- Bacillus amyloliquefaciens, T6- Trichoderma harzianum. The in vitro test was done using a dual culture technique,
while the in vivo test was done through a detached pod test. The dual culture test showed that T. harzianum reduced P.
palmivora radial growth to 6.77mm with a percent growth inhibition of 70.54%. Moreover, the detached pod test recorded
no disease incidence, severity, and incubation period in pods treated with T. harzianum. Whereas, the longer incubation
period was noted in pods treated with chemical control at 4.27 days. Moreover, the highest degree of control was recorded
in pods treated with T. harzianum (100%). These findings emphasized the potential of T. harzianum as a sustainable and
effective solution for combating cacao black pod rot and promoting sustainable agriculture.
Keywords
Phytophthora palmivora, Biological control agents, Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum
INTRODUCTION
The cacao tree is scientifically known as Theobroma cacao. It is a small (13 to 26 ft) evergreen tree in the family
Malvaceae [20] native to the deep tropical regions of Central and South America. Its seeds, cocoa beans, are used to make
cocoa mass, cocoa powder, and chocolate. The fruit or cocoa pod is an ovoid shape, 15 to 30 cm long and 8 to 10 cm
wide, ripening yellow to orange and weighing about 500 g when ripe. The pod contains 20 to 60 seeds, usually called
"beans," embedded in a white pulp. The seeds are the main ingredients of chocolate, while the pulp is used in some
countries to prepare refreshing juice, smoothies, jelly, and natal [32]. Each seed contains a significant amount of fat (40 to
50%), referred to as cocoa butter. Their most noted active constituent is theobromine, a compound similar to caffeine.
Cacao is being cultivated on 12.10 million hectares worldwide [4], with the demand for cacao increasing up to 3 % per
year [7].
In the Philippines, around 10,000 to 15,000 cacao farmers are producing cacao nationwide. Data from the
Department of Agriculture-High Value Crops Development Program (DA-HVCDP) revealed that Mindanao contributes
90 percent of the country's total cacao production, and 80 percent of which comes from the Davao Region [8].
DOI

3. 432
According to the Philippine Cacao Industry Roadmap [28], cacao may significantly contribute to poverty alleviation and
inclusive growth through livelihood and job generation because cacao production only requires small monetary
investment or start-up capital. This explains why 90% of the growers are of small farm holdings. Its diversified usage as
food and non-food warrants a sustainable marketing opportunity. At present, Philippine cacao production stands at
10,000–12,000 metric tons from the 20,000–25,000 hectares of land planted with cacao per industry estimate.
However, cacao black pod rot is a particularly economically serious problem in all cacao-producing regions
caused by Phytophthora palmivora, which causes global yield loss of 20-30% and tree deaths of 10% annually [19]. In
Central and West Africa, cacao black pod disease is caused by two species of Phytophthora, P. palmivora and P.
megakarya. If left unmanaged, it has the potential to result in crop losses ranging from 50 to 100%. Currently, multiple
control measures are being developed and implemented in order to effectively manage the condition. Systemic fungicides
are applied in regular intervals of 3 to 4 weeks. The sustainability of fungicide usage becomes problematic due to the
persistence of soil-borne Phytophthora species, which can survive in soil and infected debris for extended periods ranging
from months to several years [10, 29]. This pathogen is responsible for root infections and serves as a reservoir of
inoculum, releasing zoospores that have the potential to infect other plant parts through the transfer of water splashes
from the soil to the foliage. Furthermore, in addition to the aforementioned issues of exorbitant expenses, ecological
degradation, and concerns regarding the quality of cocoa beans. It is recommended to implement cultural practices, such
as appropriate spacing and pruning techniques, to optimize shading and aeration. These methods aim to minimize surface
wetness and thereby diminish the occurrence of the disease. Regular and thorough harvesting, cleaning, and proper
disposal of pod mummies/husks and sick pods have demonstrated some effectiveness in decreasing the presence of
secondary inoculum. Nevertheless, these approaches exhibit a high degree of labor intensity, substantial financial costs,
and economic feasibility only contingent upon the presence of elevated market prices for cocoa [10].
With the advent of RA 10068, also known as the Organic Agriculture Act of 2010, the state is searching for and
encouraging sustainable agriculture. Also, the heavy application of chemical fungicides would eventually lead to resistant
pathogens and cause soil and water pollution. The more sustainable and environmentally friendly methods should be
established and implemented, such as biological control and natural inputs.
Moreover, with the increasing awareness of environmental risks and the financial burdens associated with the use
of chemical treatments for disease control, there is a rising shift towards the exploration of biological control as a more
ecologically sustainable approach to managing plant diseases [9].
Hence, this study was conducted to evaluate the use and effectiveness of different biological control agents as an
alternative control measure of black pod rot disease in cacao.
MATERIALS AND METHODS
This study was conducted at the Crop Science Laboratory of Davao de Oro State College, Compostela, Davao de Oro,
Philippines. The experiment was laid out using a Complete Randomized Design (CRD) with six treatments and replicated
three times (Table 1).
Table 1 Treatments and rates used in the experiment
Treatment No. Treatment Rate
T1 Negative Control (Uninoculated) Untreated
T2 P. palmivora Untreated
T3 Chemical Control
(Trifloxystrobin and Tebuconazole)
0.75g/1L
T4 Bacillus subtilis 1.25g/1L
T5 Bacillus amyloliquefaciens 0.05g/1L
T6 Trichoderma harzianum 1.25g/1L
In Vitro Test for Antagonism
The Potato Dextrose Agar (PDA) medium was prepared following the standard procedure and sterilized in an autoclave at
121.6°C for 15 minutes. The sterilized PDA medium was dispensed to the petri dish by 20ml each dish.
Meanwhile, P. palmivora was isolated using a soil baiting technique where soil near the diseased plant infected
by the pathogen was collected. Soil baiting with plant material susceptible to P. palmivora helped ensure the isolation of
this specific pathogen. It provided a targeted approach for detecting the presence of this particular oomycete in soil.
For slide culture, it is a rapid method of preparing fungal growth for examination and identification. It permits P.
palmivora to be studied visually in situ with as little disturbance. Through this technique, P. palmivora are identified by
close examination of their morphology and characteristics. The P. palmivora grows directly on the slide with small
cuttings of infected leaves. By doing this, the P. palmivora structures were directly viewed under a microscope (Biobase
Model BMB-117M – Made from China). The distinct P. palmivora morphological characteristics, such as papillate
sporangium, terminal chlamydospore, and intercalary chlamydospore, were observed.
Moreover, B. subtilis and B. amyloliquefasciens are commercially available and were purchased from a legitimate
supplier. At the same time, T. harzianum was obtained from the Provincial Agriculturists Office of Davao de Oro,
Philippines.

4. 433
The assay for antagonism was performed on a PDA medium using the dual culture method [13]. Mycelial discs (3cm
diameter) of seven-day-old P. palmivora and T. harzianum while three-day-old B. subtilis and B. amyloliquefaciens were
placed on the same dish 1.5cm from each other. The mycelial disc of P. palmivora and T. harzianum were obtained from
the prepared seven-day-old pure culture while the bacterial biocon (B. subtilis and B. amyloliquefaciens) were obtained
from the prepared three-day-old pure culture. Meanwhile, the chemical control was prepared using the filter paper method
at the recommended rate of the fungicide. The fungicide (Trifloxystrobin and Tebuconazole) used was a systemic broad-
spectrum fungicide with protective and curative action that is locally available and widely used by the farmers in the
locality [29].
𝑃𝐺𝐼 (%) =
𝑅𝑎𝑑𝑖𝑎𝑙 𝐺𝑟𝑜𝑤𝑡ℎ 𝑃. 𝑝𝑎𝑙𝑚𝑖𝑣𝑜𝑟𝑎 𝑖𝑛 𝑡ℎ𝑒
𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑑𝑖𝑠ℎ − 𝑅𝑎𝑑𝑖𝑎𝑙 𝐺𝑟𝑜𝑤𝑡ℎ 𝑜𝑓𝑃. 𝑝𝑎𝑙𝑚𝑖𝑣𝑜𝑟𝑎 𝑖𝑛 𝑡ℎ𝑒
𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑑𝑖𝑠ℎ
𝑅𝑎𝑑𝑖𝑎𝑙 𝐺𝑟𝑜𝑤𝑡ℎ 𝑜𝑓 𝑃. 𝑝𝑎𝑙𝑚𝑖𝑣𝑜𝑟𝑎 𝑖𝑛 𝑡ℎ𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑑𝑖𝑠ℎ
𝑥 100
The Percent Growth Inhibition (PGI) was categorized on a growth inhibition category (GIC) scale from 0 to 4, Where: 0=
no growth inhibition: 1= 1-25% growth inhibition: 2 = 26-50% growth inhibition: and 4 = 76-100% growth inhibition.
Detached Pod Test
The source of cacao pods with the variety of "UF-18" was procured at Terec cacao farm P-1 Cabinuangan, New Bataan,
Davao de Oro, Philippines.
For the Detached Pod Test (DPT), matured unripe pods of UF18 with similar size at four (4) months old were
used as test samples. Pods were harvested with care and kept in clean sacks. The harvested cacao pods were washed
thoroughly with tap water and blotted dry. Afterward, it was surface sterilized with 10% sodium hypochlorite.
Treatments were calibrated to determine the amount of solution to be applied to the cacao pods. Proper tagging of the
sample pods was also properly done. Cacao pods were wounded using a cork borer (6mm diameter) before treatment
application. The chemical and biocon treatments were prepared using the recommended rate and applied to the cacao
pods with 0.5ml treatment solution on the wounded part per pod and air-dried for ten minutes as a preventive control.
A 14-day-old P. palmivora culture was used for the inoculation. It was standardized using a cork borer (10mm diameter),
and the mycelial discs were then inoculated to the wounded cacao pods [21]. Cacao pods were incubated in a humid
chamber using clean plastic cellophane for seven days at 28 to 32o
C.
Incidence of cacao pod rot was recorded by observing it daily for its symptom development, such as necrotic
lesions with brown or black color, which eventually enlarged to cover the whole pod. This was taken from 15 cacao
sample pods. It was computed using the formula below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 (%𝐷𝐼) =
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐼𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑓𝑟𝑢𝑖𝑡𝑠
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑟𝑢𝑖𝑡𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑
𝑥 100
The disease severity was evaluated based on five sample pods per treatment per replication. The percentage of disease
severity was determined following the damage rating scale of Akrofi [1].
Table 2 Cacao black pod rot severity rating scale
Score Infected Portion Percentage Infection Inference
0 None 0% Healthy
1 1/5 20% Not Severe
2 2/5 40% Mildly Severe
3 3/5 60% Averagely Severe
4 4/5 80% Severe
5 All 100% Extremely Severe
Based on the rating of the severity of cacao pod lesion manifest by P. palmivora, the disease index for each of the
symptoms was computed using the formula below:
𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝑆𝑒𝑣𝑒𝑟𝑖𝑡𝑦 𝐼𝑛𝑑𝑒𝑥 (𝐷𝑆𝐼) =
∑(𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑛 𝑠𝑐𝑎𝑙𝑒 𝑥 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑜𝑑𝑠 𝑖𝑛 𝑡ℎ𝑎𝑡 𝑠𝑐𝑎𝑙𝑒)
∑(𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑝𝑜𝑑𝑠)
The degree of effectiveness of the treatments was based on computed percent degree of control (%DC) using the
following arbitrary scale shown in Table 3.
Table 3 Degree of effectiveness of treatments
Disease Index (%) Degree of Effectiveness
1-20%
Not Effective
21-40% Less Effective
41-60% Moderately Effective (ME)
61-80% Effective
81-100% Very Effective

5. 434
The degree of control was computed using the formula below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑜𝑓 𝑑𝑒𝑔𝑟𝑒𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 =
% 𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 − % 𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑡𝑟𝑒𝑎𝑡𝑒𝑑
% 𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑
𝑥 100
The data from the in vitro test for antagonism and detached pod test (DPT) were analyzed using the Analysis of Variance
(ANOVA) following Completely Randomized Design (CRD). The differences among treatment means were computed
using Tukey's HSD when the variance was significant. Photo documentation was taken to support the experiment.
RESULTS AND DISCUSSION
Growth Response of P. palmivora to Different Biological Control Agents
The different biological control agents (BCAs) were tested against P. palmivora using the dual culture method. Radial
growth and percent inhibition of P. palmivora were recorded. Results showed that all three BCAs tested in this study
exhibited antagonistic activities against P. palmivora, the pathogen of cacao pod rot. All of the test antagonists
considerably hindered the radial growth of the pathogen under the conditions of this study. The T. harzianum shows the
shortest radial growth of P. palmivora with 6.77mm, followed by Bacillus subtilis with 10.33mm, then the chemical
control with 18.77mm. Extensive radial growth of P. palmivora was noted against B. amyloliquefaciens with 21.20mm
(Table 4). The results implied the potential biocontrol of T. harzianum, for it was able to inhibit the growth of P.
palmivora and subsequently reduce its radial growth due to its modes of action such as competition and antagonism with
the pathogen, production of certain compounds against pathogenic fungi and parasitism on pathogenic fungi [26]. Also, in
the study of Sarria et al. [30], T. harzianum showed other mechanisms to reduce the growth of P. palmivora, such as
mycoparasitism, competition, and production of antifungal compounds and metabolites.
Table 4 Radial growth (mm) and percent growth inhibition of P. palmivora as affected by different biological control agents
Treatments
Radial Growth of P.
palmivora (mm)1 Growth Inhibition (%)1 Growth Inhibition
Category2
P.palmivora 22.97d
- -
Chemical control 18.77c
18.30c
1
Bacillus subtilis 10.33b
55.01b
3
Bacillus amyloliquefaciens 21.20cd
7.71c
1
Trichoderma harzianum 6.77a
70.54a
3
CV (%) 6.67 13.31
1
Means with the same letter superscript are not significantly different at 1% level by Tukey’s HSD.
2
Growth Inhibition Category (GIC) scale: - = not applicable 0= no growth inhibition; 1= 1-25% growth inhibition; 2
= 26-50% growth inhibition; 3= 51-75 growth inhibition; and 4 = 76-100% growth inhibition
Moreover, the percent growth inhibition of P. palmivora followed the same trend, such in radial growth where the highest
inhibition was noted in T. harzianum with 70.54%, followed by B. subtilis with 55.01% inhibition next is chemical
control with 18.30% growth inhibition. Lastly, lesser inhibition of P. palmivora was recorded in B. amyloliquefaciens
with 7.71%.
Fig. 1 Growth response of Phytophthora palmivora (Right) to biological control agents (Left); A.) Chemical control; B.) Bacillus
subtilis; C.) Bacillus amyloliquefaciens; D.) Trichoderma harzianum
According to Fatima et al. [12], T. harzianum was found to be an effective inhibitor reduction in the radial growth of
Phytophthora infestans with an inhibition rate of (85%). Based on Moayedi and Ghalamfarsa’s [24] report, the isolation
of mycoparasitic Trichoderma can suppress Phytophthora root rot. Pal and McSpadden [27] reported that the application
of T. harzianum produces potential antibiotics and other chemicals that are harmful to pathogens and inhibit their growth.
It produces gliotoxin, which inhibits Rhizoctonia solani, a soil-borne pathogen that causes root rot in plants. Moreover,
according to Zeidan [33], T. harzianum can control several soil-borne fungal pathogens.
D
C
B
A

6. 435
On the other hand, B. subtilis is potentially useful as a biological control agent and produces various biologically active
compounds with a broad spectrum of activities toward phytopathogens [31]. The strains of bacteria Bacillus have been
widely used against many economically important plant pathogenic fungi, possess a resistant spore stage, produce several
kinds of antifungal compounds, and have shown significant inhibitory activity against Fusarium moniliforme and
Colletotrichum gloeosporioides [17]. Thus, through their various mechanisms against the pathogen, this might be one of
the reasons that T. harzianum and B. subtilis got the highest growth inhibition of P. palmivora.
Detached Pod Test
The detached pod test was conducted to validate the result of the in vitro test. The same biological control agents were
used in this test, such as B. subtilis, B. amyloliquefaciens, and T. harzianum. Different BCAs were applied on four-month-
old UF18 cacao pods prior to the inoculation of P. palmivora. The data, such as disease incidence, incubation period, and
severity of infection, were evaluated after seven days of P. palmivora inoculation. Based on the results, cacao pods
treated with T. harzianum recorded no disease incidence (0%), followed by B. amyloliquefaciens and B. subtilis with 40%
and 13.33% disease incidence, respectively. The cacao pods treated with a fungicide (chemical control) had a disease
incidence of 86.67%, while the untreated cacao pods inoculated with P. palmivora got 100% disease incidence (Table 5).
The efficacy of biocontrol agents was effectively established using the detached pod test, which demonstrated the
potential of T. harzianum as a highly effective solution with a disease incidence of 0%.
Table 5 Summary of the data gathered on detached pod test inoculated with P. palmivora
Treatments
Disease Incidence
(%)**/1
Incubation Period
(days)**
Disease Severity on
cacao pods**
Negative Control 0.00a
0.00c
0.00a
P. palmivora 100.00d
3.73a
1.00c
Chemical control 86.67cd
4.27a
0.87c
Bacillus subtilis 13.33ab
1.27c
0.20ab
Bacillus amyloliquefaciens 40.00bc
2.40ab
0.40b
Trichoderma harzianum 0.00a
0.00c
0.00a
CV (%) 24.59 40.38 34.49
1
Data were Square root transformed prior to analysis of variance.
**Means with the same letter superscript are not significantly different at 1% level by Tukey’s HSD
Moreover, the symptom appearance of black pod rot on inoculated cacao pods treated with B. amyloliquefaciens is 2.40
days, followed by B. subtilis with 1.27 days, while no symptom appearance on cacao treated with T. harzianum.
Meanwhile, cacao pods treated with chemical control got the longest symptom appearance at 4.27 days, followed by the
untreated and inoculated cacao pods at 3.73 days. The recent research conducted by Liu et al. [22] further reinforces this
by highlighting Trichoderma's ability to achieve a 0% disease incidence, underscoring its exceptional biocontrol potential.
These mechanisms encompass mycoparasitism, antibiosis, and competition for resources, ultimately enabling
Trichoderma to outcompete and suppress the growth of oomycete pathogens like P. palmivora [6, 11]. Moreover,
Trichoderma species are renowned for their capacity to colonize plant roots and induce systemic resistance, thereby
enhancing their biocontrol capabilities [18]. The consistency of Trichoderma's effectiveness across recent studies
underscores its potential as a sustainable and environmentally friendly solution for disease management in cacao
production.
Fig. 2 Cacao pods treated with different biological control agents after seven days of inoculation: A.) Sterilized Distilled Water (SDW); B.)
Phytophthora palmivora; C.) Chemical control; D.) Bacillus subtilis; E.) Bacillus amyloliquefaciens; F.) Trichoderma harzianum
A B
C D
E F

7. 436
Degree of Control
Results showed that T. harzianum got the rating of very effective due to zero disease incidence recorded, and B. subtilis
with a rating of very effective with 13.33% disease incidence, followed by B. amyloliquefaciens with a moderately
effective rating with 40% disease incidence. Whereas chemical control was rated as not effective due to a high pod rot
incidence of 86.67%. However, the highest disease incidence was noted on positive control (inoculated and untreated)
with 100% disease incidence (Table 6).
Table 6 Percentage of disease incidence and degree of control of black pod rot on cacao treated with
different biological control agents1
Treatments
Disease
Incidence**/1
Degree of
control
Degree of effectiveness
Sterilize Distilled Water (SDW) 0.00a
- -
P. palmivora 100.00d
- -
Chemical control 86.67cd
13.33b
Not Effective
Bacillus subtilis 13.33ab
86.67a
Very Effective
Bacillus amyloliquefaciens 40.00bc
60.00a
Moderately Effective
Trichoderma harzianum 0.00a
100.00a
Very Effective
CV %= 24.59
1
Data subjected to ANOVA were Square root transformed.
** Means with the same letter superscript are not significantly different at 1% level by Tukey’s HSD.
Based on the result, it is evident that T. harzianum is very effective among the different biological control agents in
managing black pod rot on cacao pods. According to Claydon et al. [5], T. harzianum was able to reduce disease
incidence of the pathogen. Meanwhile, based on the report of Mpika et al. [25], T. harzianum was tested, and the inhibitor
effect of the P. palmivora agent of brown rot cacao pods of 83.33%. The result implied that T. harzianum is a potential
antagonist against P. palmivora, causing black pod rot of cacao [15]. Trichoderma employs various mechanisms that
reduce the incidence of plant diseases. These mechanisms encompass nutrient and space competition, synthesis of
antifungal metabolites, mycoparasitism, production of lytic enzymes that degrade cell walls of fungal plant pathogens,
and the induction of plant resistance [3, 14]. In the study of Mahadevi et al. [23], T. harziaum showed potential biocontrol
against Phytophthora root rot disease in Papaya caused by P. palmivora
Meanwhile, B. subtilis has both a direct and indirect biocontrol mechanism for suppressing pathogen-caused
disease. The direct mechanism involves the synthesis of numerous secondary metabolites, hormones, cell wall-degrading
enzymes, and antioxidants, all of which aid the plant's defense against pathogen attack. The indirect mechanism includes
plant growth stimulation and the induction of acquired systemic resistance [16]. In the study by Ashwini and Srividya [2],
B. subtilis showed a 65% reduction in disease incidence caused by Colletotrichum gloeosporioides and Phytophthora
capsici.
CONCLUSION
Different biological control agents were tested against cacao black pod rot disease. The biological control agents used in
the study were B. subtilis, B. amyloliquefaciens, and T. harzianum. The dual culture test showed that T. harzianum
reduced P. palmivora radial growth to 6.77mm with a percent growth inhibition of 70.54%. Moreover, the detached pod
test recorded no disease incidence (0%), severity, and incubation period in pods treated with T. harzianum. Whereas, the
longer incubation period was noted in chemical control of 4.27 days. Moreover, the highest degree of control was
recorded in pods treated with T. harzianum (100.00%).
Therefore, based on the result of the study, the application of T. harzianum can potentially control the cacao black
pod rot caused by P. palmivora in both dual culture and detached pod tests. It is recommended to further study the
application of T. harzianum on the cacao field for validation of the result.
FUNDING INFORMATION
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
DECLARATION OF CONFLICT
The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
ACKNOWLEDGMENTS
The authors would like to thank the Davao de Oro State College (DDOSC) for allowing the experiment to be conducted
in the Crop Science Laboratory and providing the necessary laboratory equipment. Also, to Mr. Jupite Mark U. Banayag
and Mr. Lemuel L. Alvior, Jr. for the guidance and shared expertise. Likewise, to Mr. Mariano Coraje for his assistance
during the implementation of the study.

8. 437
REFERENCES
1. Akrofi, A. Y. (2015). Phytophthora megakarya: A Review on its Status as a Pathogen on Cacao in West Africa. African
Crop Science Journal, Vol. 23, No. 1, pp.67-87
2. Ashwini N., & Srividya S. (2013). Potentiality of Bacillus subtilis as a biocontrol agent for the management of anthracnose
disease of chili caused by Colletotrichum gloeosporioides OGCI.
3. Asad, S. A. (2022). Mechanisms of action and biocontrol potential of Trichoderma against fungal plant diseases - A review.
Ecological Complexity, 49, 100978. https://doi.org/10.1016/j.ecocom.2021.100978
4. Bermudez, S., Voora, V., Larrea, C., & Luna, E. (2022). Cacao Prices and Sustainability. Global Market Report.
5. Claydon N., Allan M., Hanson J. R., & Avent A. G. (1987). Antifungal alkyl pyrones of Trichoderma harzianum.
Transaction of the british Mycological Society 88, 503-13.
6. Contreras-Cornejo, H. A., Macías-Rodríguez, L., & Cortés-Penagos, C. (2016). Mechanisms and practical considerations
involved in plant growth promotion by rhizobacteria. Journal of Soil Science and Plant Nutrition, 16(4), 1-18.
7. DA-HVCDP XI (2015). Department of Agriculture XI- High Value Crops Development Program. Cacao Production Guide
(Cebuano).
8. DA-HVCDP XI (2016). Department of Agriculture XI- High Value Crops Development Program. Cacao Production Guide
(Cebuano). No. 01-B Revised June 2016.
9. Deberdt P., Mfegue C.V., Tondje P.R., Bon M.C. Ducamp M., Hurard C., Begoude B.A.D., Ndoumbe-Nkeng M., Hebbar
P.K.,& Cilas C. (2008). Impact of environmental factors, chemical fungicide and biological control on cacao pod production
dynamics and black pod disease (Phytophthora megakarya) in Cameroon, Biol. Control 44:149–159.
10. Doungous, O., Emile, M., Longue, E. a. M., & Nkengafac, N. J. (2018). Potentials of cocoa pod husk-based compost on
Phytophthora pod rot disease suppression, soil fertility, and Theobroma cacao L. growth. Environmental Science and
Pollution Research, 25(25), 25327–25335. https://doi.org/10.1007/s11356-018-2591-0
11. Druzhinina, I. S., Seidl-Seiboth, V., Herrera-Estrella, A., Horwitz, B. A., Kenerley, C. M., Monte, E., & Kubicek, C. P.
(2011). Trichoderma: the genomics of opportunistic success. Nature Reviews Microbiology, 9(10), 749-759.
12. Fatima, K., Noureddine, K., Henni, J.E., Mabrouk, K. (2015). Antagonistic effect of Trichoderma harzianum against
Phytophthora infestans-west of Algeria, Vol. 6, No. 4, p. 44-53, 2015.
13. Fokkema, N.J. (1978). Fungal antagonism in the phylosphere. Ann. Appl. Biol. 89, 115-117.
14. Ghorbanpour, M., Omidvari, M., Abbaszadeh-Dahaji, P., Omidvar, R., & Kariman, K. (2018). Mechanisms underlying the
protective effects of beneficial fungi against plant diseases. Biological Control, 117, 147-157.
15. Haran, S., Schickler, H., Oppencheim, A., & Chet I. (1996). Different expression of Trichoderma harzianum chitinases
during mycoparasitism. Phytopathology, 86, 980-985
16. Hashem, A., Tabassum, B., & Abd_Allah, E. F. (2019). Bacillus subtilis: A plant-growth-promoting rhizobacterium that also
impacts biotic stress. Saudi Journal of Biological Sciences, 26(6), 1291–1297. https://doi.org/10.1016/j.sjbs.2019.05.004
17. Havenga., W., De Jager, E. S., & Korsten, L. (1999). Factors affecting biocontrol efficacy of Bacillus subtilis against
Colletotrichum gloeosporioides. S. Afr. Avocado Growers Assoc. Year, 22, 12-20.
18. Hermosa, R., Viterbo, A., Chet, I., & Monte, E. (2012). Plant-beneficial effects of Trichoderma and of its genes.
Microbiology, 158(1), 17-25.
19. International Cocoa Organization (ICCO). (2015). The Chocolate Industry. ICCO. Retrieved from http://www.icco.
org/about-cocoa/chocolate-industry.html
20. Juan C., Motamayor P. L., Wi, Da Silv, J., Reg, L., David, N., Steven, B., & Raymond J. (2008). Geographic and Genetic
Population Differentiation of the Amazonian Chocolate Tree (Theobroma cacao) PLoS ONE 3(10):1371-1376.
21. Kudjordjie, E. (2015). Phytophthora megakarya and P. palmivora on Theobroma cacao: Aspects of Virulence and The
Effects of Temperature on Growth and Resistance to Fungicides. Journal of Plant Pathology 11-23.
22. Liu, Y., He, P., He, P., Munir, S., Ahmed, A., Wu, Y., Yang, Y., Lu, J., Wang, J., Yang, J., Pan, X., & Tian, Y. (2022).
Potential biocontrol efficiency of Trichoderma species against oomycete pathogens. Frontiers in Microbiology, Volume 13 -
2022, https://doi.org/10.3389/fmicb.2022.974024.
23. Mahadevi, M., Tahavaselvi, A., Latha, V., & Panneerselvam, A. (2018). Invitro screening and molecular characterization of
Trichoderma harzianum against Phytophthora palmivora (Butl.,) causing root rot disease in Caricapapaya L. International
Journal of Scientific Research in Science and Technology, 4(5), 156-168
24. Moayedi, G., & Mosstowfizadeh-Ghalamfarsa, R. (2010). Antagonistic Activities of Trichoderma spp. on Phytophthora
Root Rot of Sugar Beet. Iran Agriculture Research 28(2), and 29, 1-2.
25. Mpika J., Kebe I.B., Druzhinina I.S.S., Komon-Zelazowska M., Kubicek C.P., & Ake S. (2009). Inhibition of Phytophthora
palmivora, agent of cacao pod rot, Trichodderma sp. Science and Nature Vol. 6 (1), 49-62.
26. Niogret J, Sanchez, V., & Marelli. J. (2020). Increasing the resilience of cacao to major pest and disease threats in the 21st
century, proceedings of an Asia-Pacific Regional Cocoa IPM symposium, ACIAR Proceedings Series, No. 149, Australian
Centre for International Agricultural Research, Canberra, 97 pp.
27. Pal, K., & McSpadden, G. (2006). Biological Control of Plant Pathogens. The Plant Health Instructor. P1-25.
28. Philippine Cacao Industry. Philippine Cacao Philippine Cacao Industry Roadmap 2017-2018. Philippine CacaoIndustry
Roadmap. (2017). Retrieved from
http://bpi.da.gov.ph/bpi/images/PDF_file/Cacao%20Industry%20Roadmap%20%20Signed%20%20%20March%2010,%202
017.pdf.
29. Riedel, J., Kägi, N., Armengot, L., & Schneider, M. (2019). Effects of rehabilitation pruning and agroforestry on cacao tree
development and yield in an older full-sun plantation. Experimental Agriculture, 55(6), 849–865.
https://doi.org/10.1017/s0014479718000431
30. Sarria, G., García, A., Mestizo, Y. A., Medina, C. L., Varón, F., Mesa, E., & Hernández, S. (2021). Antagonistic Interactions
Between Trichoderma spp. and Phytophthora palmivora (Butler) From Oil Palm In Colombia. European Journal of Plant
Pathology, 161(4), 751–768. https://doi.org/10.1007/s10658-021-02363-z

9. 438
31. Stein, T. (2005). Bacillus subtilis antibiotics: structures, syntheses, and specific functions. Molecular Microbiology 56, no. 4:
845-857.
32. Terry, G., Jeffery, W., Carmen, M, Ponciano, O., Michael, B., David, C., Michael, D., & Hodgson, J. (2007). Oldest
chocolate in the New World. Antiquity 81:314-318.
33. Zeidan, E. H., Saad, M. and Farage, S. (2005). Biological controls of grapevine root rot by antagonistic microorganism’s.
African Mycol Biotechnol. 13(3): 19-36.
View publication stats